Proton-coupled electron transfer (PCET) is the basic mechanism by which the energy conversion processes in a remarkable variety of oxidases and reductases are effected. Small molecule activation, redox-driven proton pumps and enzymatic function derived from hydrogen atom abstraction all involve the coupling of an electron to proton motion. By studying PCET networks in biomimetic and natural systems, the authors intend to develop a framework in which PCET-derived structure/function relations of enzymes and proteins may be defined. With the design and synthesis of new model compounds, the authors may photo-induce electron transfer (ET) within a donor/acceptor pair that has a proton transfer (PT) network internal (e.g., salt bridge) or external (e.g., imine) to the electron transfer pathway. A key to their approach is the incorporation of independent optical and/or vibrational signatures for the electron transfer and the proton transfer events so that they can monitor the fate of the proton, in response to the electron and vice versa. With this development they can define the factors that distinguish synchronous and asynchronous transfer of the electron and proton, the structural/electronic features by which the proton and electron communicate with each other and how the energetics (e.g., reorganization, free energy) cause PCET to differ from an ET reaction. Against this mechanistic backdrop, they will undertake studies to directly measure the PCET pathway in ribonucleotide reductase (RNR). In this enzyme, an oxidizing hole traverses a putative 35 A inter-subunit (R1 and R2) pathway to arrive at an active site where the reduction of ribonucleotide to deoxyribonucleotide is catalyzed. They intend to break down the overall pathway by studying PCET within the individual R2 and R1 subunits. For the latter, they will focus on the 20-mer C-terminal peptide tail (R2C20) from the R2 subunit, which accounts for the predominate interaction required for subunit association. This peptide contains a tyrosine (Y356) that is thought to shuttle the hole from R1 to R2. A major focus of this proposal is to develop general photochemical methods to trigger the release of radical amino acids along PCET pathways of proteins and enzymes. Modification of the Y356 position of R2C20 with one of these newly synthesized tyrosyl photocages will enable them to generate Y356 radical upon laser excitation, while bypassing the normal radical generating process originating at the metallo-cofactor of R2. By turning the tyrosine radical on instantly, they can observe the transport of the hole along the PCET pathway into the R1 active site by transient absorption methods and correlate this transport to effector and substrate binding at R1. The pKa's and driving force of the PCET pathway can be modified with fluorotyrosines, allowing them to assess the relative importance of the contributions of the proton and electron to the PCET. The intein/extein technology of protein splicing provides a further opportunity to introducte the R2C20 C-terminal tail back into R2 with a photocaged fluorotyrosine at Y356. In this case, they will be able to study the PCET pathway through R2 by laser-generating Y356 radical, which can then propagate backwards to the tyrosine (Y122) proximate to the diiron cofactor of R2, the site from which overall PCET is initiated in RNR.